Alexfluor 488 conjugated anti rabbit goat antibody

HEK293T cells were cultured on glass-bottom culture dishes (MatTek, P35G-0–14-C) and transfected with CoV expression constructs as previously described. Cells were fixed with 4% paraformaldehyde-PBS, washed thrice with PBS, then permeabilized in 0.2% Triton-X (in PBS). After three PBS washes, cells were blocked in PBS with 1% BSA with 0.1% Saponin (blocking buffer). After blocking, cells were incubated with anti-PDIA4 primary antibody (Protein Tech, 14712–1-AP) in blocking buffer (1:1000 dilution) for 1 hour at 37°C. After three PBS washes, cells were incubated with AlexFluor 488-conjugated anti-rabbit goat antibody (ThermoFisher, A-11008) in blocking buffer (1:500 dilution) at room temperature for 30 min. Cells were then stained with M2 FLAG primary antibody (SigmaAldrich, F1804) and AlexFluor 594-conjugated anti-mouse goat antibody (ThermoFisher, A-11005) using the same conditions. Cells were then mounted in Prolong Gold with DAPI stain (ThermoFisher, P36935). Cells were imaged using an LSM-880 confocal microscope (Zeiss) and images were merged using Image J software.

HEK293T cells were transfected with nsp3 constructs as described in “Cell culture and transfection.” Two hours post media change, 2 × 10⁴ transfected cells were seeded into glass-bottom culture dishes (MatTek, P35G-0-14-C). At 40 h posttransfection, cells were fixed with 4% paraformaldehyde-PBS, washed with PBS three times, then permeabilized in 0.2% Triton-X (in PBS). After three PBS washes, cells were blocked in PBS with 1% BSA and 0.1% Saponin (blocking buffer) for 1 h at room temperature. After blocking, cells were incubated with anti-PDIA4 primary antibody (Protein Tech, 14712-1-AP) in blocking buffer (1:1000 dilution) overnight at 4 °C. After three PBS washes, cells were incubated with AlexFluor 488-conjugated anti-rabbit goat antibody (ThermoFisher, A-11008) in blocking buffer (1:500 dilution) at room temperature for 30 min. Cells were then stained with M2 FLAG primary antibody (SigmaAldrich, F1804) and AlexFluor 594-conjugated anti-mouse goat antibody (ThermoFisher, A-11005) using the same conditions. Cells were then mounted in Prolong Gold with DAPI stain (ThermoFisher, P36935) overnight. Cells were imaged using an LSM-880 or LSM-710 confocal microscope (Zeiss), and images were merged using Image J software.